Polymerase chain reaction (PCR) following cDNA synthesis from mRNA (reverse transcription-polymerase chain reaction, RT-PCR) to analyze gene expression of any specific mRNA in cells and tissues has become common technique, because of its better sensitivity and less labor-intensive manipulations than the traditional Northern blot (Kawasaki E S, Wang A M, “Detection of gene expression. In: Erlich, E A. PCR technology”, New York: Stockton, 1989; 89-97). Furthermore, because recently available recombinant Tth thermostable polymerase has activities of both reverse transcriptase and DNA polymerase, both steps can be performed simultaneously in a single tube without changing the buffer system (Myers T W, Gelfand D H, “Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase”, Biochemistry 1991; 30:7661-6). However, it still requires purification of total RNA or mRNA from cells and tissues, which takes additional time-consuming, and labor-intensive step(s).
It has been reported that mRNA is successfully captured by an oligo(dT)immobilized polystyrene (PS) microplate (GENEPLATE®, Hitachi Chemical Co., Japan, and AGCT, Irvine, Calif.) (Mitsuhashi M, et al., “Gene manipulation on plastic plates”, Nature 1992:357:519-20, Miura Y, et al., “Fluorometric determination of total mRNA with oligo(dT) immobilized on microtiter plates”, Clin Chem 1996:42:1758-64, Miura Y, et al., “Rapid cytocidal and cytostatic chemosensitivity test by measuring total amount of mRNA”, Cancer Lett 1997:116:13944) followed by single- and double-stranded cDNA synthesis on the plate (Tominaga K, et al., “Colorimetric ELISA measurement of specific mRNA on immobilized-oligonucleotide-coated microtiter plates by reverse transcription with biotinylated mononucleotides”, Clin Chem 1996:42:1750-7). Once double stranded cDNA is formed on a PS microplate of GENEPLATE®, sense stranded cDNA can be removed and used as a template for multiple PCR experiments (Ishikawa T, et al., “Construction of cDNA bank from biopsy specimens for multiple gene analysis of cancer”, Clin Chem 1997:43:764-70). Unfortunately, PCR cannot be performed on this PS microplate, because of its heat instability during the denaturing step in PCR cycles. Although heat stable polypropylene (PP) tubes and microplates are primary vessels for PCR, it is difficult to immobilize oligonucleotides onto a PP surface, because of its extremely chemically stable surface characteristics. However, oligo(dT)-immobilized polypropylene plates have recently become available.
As described above, RT-PCB is a very useful technology in various fields including diagnostic molecular pathology (Bortolin S, et al., “Quantitative RT-PCR combined with time-resolved fluorometry for determination of BCR-ABL mRNA”, Clin Chem 1996:42:1924-9). However, there are many steps involved in the analysis of RT-PCR; collection of cells, purification of RNA/mRNA, cDNA synthesis, PCR, and quantitation of PCR products. In particular, the purification of intact RNA molecules is the critical first step for the successful RT-PCR, and this requires labor-intensive multiple manipulations to eliminate or inactivate endogenous or contaminated ribonuclease (RNase) in cells and tissues. Although recent PCR technologies allow researchers to continuously monitor the quantity of PCR products with various in-line or off-line verification procedures of amplified PCR products (Morris T, et al., “Rapid reverse transcription-PCR detection of hepatitis C virus RNA In serum by using the TaqMan fluorogenic detection system”, J Clin Microbiol 1996:34:p2933-6, Wittwer C T, et al., “The LightCycler: A microvolume multisample fluorimeter with rapid temperature control”, BioTechniques 1997:22:171-181), lack of a simplified RNA preparation system prevents full automation of RT-PCR.